The goals of our studies are to understand the mechanisms by which alphaherpesviruses spread from cell-to-cell in the infected host. The alphaherpesviruses include the human pathogens herpes simplex virus (HSV) types 1 and 2 and varicella-zoster virus (VZV). Infection with these viruses is highly prevalent. These viruses are responsible for a variety of human diseases that range from trivial to life threatening. Other alphaherpesviruses include the swine pathogen, pseudorabies virus (PRV), and numerous viruses of livestock that are responsible for significant economic losses in the agricultural industry. Transmission of virus from cell-to-cell represents the major route by which alphaherpesviruses spread in their hosts. We have identified new requirements for the spread of these viruses between cells. A molecular dissection of this process will lead to a clearer understanding of the biology of these important human and animal pathogens and identify new targets for antiviral intervention. The identification of gene products that participate in cell-to-cell spread represents a major step towards these goals. To do this we will take advantage of newly isolated mutant viruses and recently identified cell lines that display novel phenotypes relating to cell-to-cell spread. Our studies will focus on PRV, a well-studied swine pathogen. PRV is closely related to HSV-1, HSV-2 and VZV, and offers several important advantages for studying alphaherpesvirus biology over its human counterparts. We have found that insertions in the glycoproteins gG locus of certain PRV strains markedly affects the ability of virus to spread from cell-to-cell in a cell-type specific manner. Our preliminary experiments have shown that elimination of gG expression does not result in this phenotype. It is our hypothesis that insertions in the gG locus affect the expression of an upstream gene, Us3, which utilizes a polyadenylation site located downstream of the gG gene. The Us3 gene encodes a protein kinase. The goal of Specific Aim 1 is to define the role of Us3 in cell-to-cell spread. Specific Aims 2 and 3 focus on genetic approaches for the identification of viral genes that can compensate for insertions in the gG locus. Our hypothesis is that these molecules will also participate in the spread of virus from cell-to-cell.